Synthetic substrates specific to activated plasmin can monitor the enzymatic functional status in situ in breast cancer cells

Chem Biol Drug Des. 2014 Jan;83(1):52-7. doi: 10.1111/cbdd.12232. Epub 2013 Oct 30.


We here strove to overcome the limitations of expression analyses such as PCR and IHC, based on molecular recognition between target and probe molecules, by designing synthetic substrates specific to the target molecules to directly estimate the enzymatic functionality in situ. The specific substrate contains a probing unit, which is an organic fragment for specific enzyme binding, and a reactive unit, which is a natural peptide subject to catalysis. In this study, the activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells using the plasmin-specific synthetic substrates designed from their inhibitors. The localization and function of the activated plasmin were successfully visualized by fluorophore combined with the specific substrate concurrently. This would be the first time for activated plasmin at work in situ by direct observation. Our concept to directly monitor the functionality of target enzymes can be used straightforwardly for other proteases such as cathepsins or caspases. Also, this substrate concept as a 'tailor-made substrate' would be utilized as a novel functional molecular probe in vivo with appropriate detectable probes.

Keywords: breast cancer; imaging probes; in situ functionality; plasminogen-plasmin system; specific synthetic substrates; structure-based molecular design.

Publication types

  • Letter

MeSH terms

  • Binding Sites
  • Breast Neoplasms / enzymology
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Line, Tumor
  • Female
  • Fibrinolysin / antagonists & inhibitors
  • Fibrinolysin / metabolism*
  • Fluorescent Dyes / chemistry
  • Humans
  • Kinetics
  • Microscopy, Confocal
  • Molecular Docking Simulation
  • Plasminogen / metabolism
  • Protease Inhibitors / chemical synthesis*
  • Protease Inhibitors / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Substrate Specificity


  • Fluorescent Dyes
  • Protease Inhibitors
  • Plasminogen
  • Fibrinolysin